棉花TRIPTYCHON基因的电子克隆和功能初步验证
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摘要
棉花纤维的长度是评价棉花纤维内在品质的一个很重要的指标,同时单粒籽棉的棉花纤维数量直接影响棉花皮棉产量。
研究表明,植物表皮细胞的发育受多个基因调控,表皮细胞的发育在不同植物中具有相似的基因调控模式。棉花纤维是胚珠外珠被表皮细胞发育延伸而成的表皮毛,棉花纤维细胞的发育与拟南芥表皮毛发育模式相似。拟南芥中存在GLABROUS1 (GL1)、GLABRA2 (GL2)、GLABRA3 (GL3)、TRANSPARENT TESTA GLABRA1 (TTG1)、TRANSPARENT TESTA GLABRA2 (TTG2)等基因促进拟南芥表皮毛的发育,同时又存在TRIPTYCHON(TRY)和CAPRICE (CPC)基因抑制拟南芥表皮毛的发育。前人研究报道,棉花中也存在TTG1、GL1、GL2等同源基因促进棉纤维的发育,但是棉花中尚未报道是否存在抑制棉花纤维发育的与TRY和CPC同源基因。
本研究利用电子克隆和RT-PCR相结合的方法从徐州142无绒无絮突变体棉花中克隆到棉花TRY基因。经过RT-PCR克隆后测序,棉花TRY基因ORF(Open read frame)全长270bp,编码89个氨基酸,其蛋白质由18种氨基酸组成,其基因实测序列的ORF碱基序列与电子克隆的ORF的碱基序列仅有一个位点的差异,各自的ORF翻译为蛋白后,其氨基酸序列仅有一个位点的差异,表明电子克隆所得基因序列具有很强的真实性。
利用生物信息学分析,该蛋白分子量为10638.1 Da;理论等电点pI:8.66;原子组成是C461H734N140O136S7;消光系数280nm :19605;不稳定系数是75.26;脂肪系数是61.35;总平均亲水性为-0.899。同拟南芥TRY蛋白一样,不存在信号肽、糖基化位点、跨膜结构、卷曲螺旋结构。同拟南芥TRY蛋白一样定位于细胞核中,同属于不稳定、疏水性蛋白,MYB类转录因子。功能预测表明,棉花TRY基因编码的蛋白在细胞内部将执行同拟南芥TRY基因编码的蛋白相似的功能。
将棉花TRY基因重组到pBI121表达载体上,构建了棉花TRY基因的表达载体,同时利用农杆菌介导的方法将棉花TRY基因整合到烟草基因组中。通过实时荧光PCR检测,获得阳性转基因植株。对转基因烟草阳性植株叶毛发育情况和根毛发育情况的观察和分析,初步表明棉花TRY基因具有可以抑制表皮毛发育这一功能。
The length of cotton fiber is one important standard to evaluate the quality of cotton fibre. And the quantity of seed cotton fiber is direct factor to affect ginned cotton harvest.
The study reveals that the growth of plant epidermal cell be regulated by many genes and the development of plant epidermal cell has similar molecule regulating pattern. The study reveals that cotton fiber is epidermal hair, epidermal cell of ovule outer integument from cotton fiber. And cotton fiber cell possesses the similar molecule regulating mode as the growth of epidermal hair cell in Arabidopsis. GLABROUS1 (GL1) / GLABRA2 (GL2) / GLABRA3 (GL3) / TRANSPARENT TESTA GLABRA1 (TTG1) /TRANSPARENT TESTA GLABRA2 (TTG2) to promot development of epidermal hair and TRIPTYCHON(TRY)/CAPRICE(CPC) inhibit development of epidermal hair in Arabidopsis. The study reveals homologous genes of TTG1/GL1/GL2 to promot development of cotton fiber in cotton, but no report about homologous genes of TRY/CPC in cotton.
In silico cloning and RT-PCR technique was performed to isolate a TRIPTYCHON(TRY) gene in Xuzhou 142 no fiber mutants. After RT-PCR cloning, the DNA fragment was sequenced, and an ORF (Open read frame) of TRY which was 270bp in length, encoding 89 amino acids was found. The Protein is composed of 18 different amino acids. There are only one locus difference between the actual DNA sequence of ORF and the DNA sequence of ORF by in silico cloning. Both of their protein had only one animol acid difference, which indicated that the gene got from in silico cloning is more authentic.
Through bioinformatics analysis, the protein molecular weight is 10638 Da, and theoretical pI:8.66, atomic composition: C461H734N140O136S7, extinction coefficients measured at 280 nm: 19605, the instability index: 75.26, aliphatic index: 61.35, grand average of hydropathicity: -0.899. This protein is same as Arabidopsis TRY protein and located in nucleolus. It belongs to instability, hydrophobic proteins and transcription factors of MYB family, and without signal peptide, glycosylation sites, trans-membrane structure, and coiled coil structure. Function prediction demonstrated that the protein coded by cotton TRY gene plays the same function as the protein does, coded by Arabidopsis TRY gene.
Cotton TRY gene was recombined to pBI121 vector.. And integrate the cotton TRY gene to tobacco genome by the ways of Agrobacterium tumefaciens mediating. Masculine transgene plant could be got by real time fluorescence PCR detection. It is initially indicated that cotton TRY gene possess the function to restrain the growth of epidermal hair cell by observing and analyzing the development of Masculine transgene tobacco leaf hair and roots hair.
研究表明,植物表皮细胞的发育受多个基因调控,表皮细胞的发育在不同植物中具有相似的基因调控模式。棉花纤维是胚珠外珠被表皮细胞发育延伸而成的表皮毛,棉花纤维细胞的发育与拟南芥表皮毛发育模式相似。拟南芥中存在GLABROUS1 (GL1)、GLABRA2 (GL2)、GLABRA3 (GL3)、TRANSPARENT TESTA GLABRA1 (TTG1)、TRANSPARENT TESTA GLABRA2 (TTG2)等基因促进拟南芥表皮毛的发育,同时又存在TRIPTYCHON(TRY)和CAPRICE (CPC)基因抑制拟南芥表皮毛的发育。前人研究报道,棉花中也存在TTG1、GL1、GL2等同源基因促进棉纤维的发育,但是棉花中尚未报道是否存在抑制棉花纤维发育的与TRY和CPC同源基因。
本研究利用电子克隆和RT-PCR相结合的方法从徐州142无绒无絮突变体棉花中克隆到棉花TRY基因。经过RT-PCR克隆后测序,棉花TRY基因ORF(Open read frame)全长270bp,编码89个氨基酸,其蛋白质由18种氨基酸组成,其基因实测序列的ORF碱基序列与电子克隆的ORF的碱基序列仅有一个位点的差异,各自的ORF翻译为蛋白后,其氨基酸序列仅有一个位点的差异,表明电子克隆所得基因序列具有很强的真实性。
利用生物信息学分析,该蛋白分子量为10638.1 Da;理论等电点pI:8.66;原子组成是C461H734N140O136S7;消光系数280nm :19605;不稳定系数是75.26;脂肪系数是61.35;总平均亲水性为-0.899。同拟南芥TRY蛋白一样,不存在信号肽、糖基化位点、跨膜结构、卷曲螺旋结构。同拟南芥TRY蛋白一样定位于细胞核中,同属于不稳定、疏水性蛋白,MYB类转录因子。功能预测表明,棉花TRY基因编码的蛋白在细胞内部将执行同拟南芥TRY基因编码的蛋白相似的功能。
将棉花TRY基因重组到pBI121表达载体上,构建了棉花TRY基因的表达载体,同时利用农杆菌介导的方法将棉花TRY基因整合到烟草基因组中。通过实时荧光PCR检测,获得阳性转基因植株。对转基因烟草阳性植株叶毛发育情况和根毛发育情况的观察和分析,初步表明棉花TRY基因具有可以抑制表皮毛发育这一功能。
The length of cotton fiber is one important standard to evaluate the quality of cotton fibre. And the quantity of seed cotton fiber is direct factor to affect ginned cotton harvest.
The study reveals that the growth of plant epidermal cell be regulated by many genes and the development of plant epidermal cell has similar molecule regulating pattern. The study reveals that cotton fiber is epidermal hair, epidermal cell of ovule outer integument from cotton fiber. And cotton fiber cell possesses the similar molecule regulating mode as the growth of epidermal hair cell in Arabidopsis. GLABROUS1 (GL1) / GLABRA2 (GL2) / GLABRA3 (GL3) / TRANSPARENT TESTA GLABRA1 (TTG1) /TRANSPARENT TESTA GLABRA2 (TTG2) to promot development of epidermal hair and TRIPTYCHON(TRY)/CAPRICE(CPC) inhibit development of epidermal hair in Arabidopsis. The study reveals homologous genes of TTG1/GL1/GL2 to promot development of cotton fiber in cotton, but no report about homologous genes of TRY/CPC in cotton.
In silico cloning and RT-PCR technique was performed to isolate a TRIPTYCHON(TRY) gene in Xuzhou 142 no fiber mutants. After RT-PCR cloning, the DNA fragment was sequenced, and an ORF (Open read frame) of TRY which was 270bp in length, encoding 89 amino acids was found. The Protein is composed of 18 different amino acids. There are only one locus difference between the actual DNA sequence of ORF and the DNA sequence of ORF by in silico cloning. Both of their protein had only one animol acid difference, which indicated that the gene got from in silico cloning is more authentic.
Through bioinformatics analysis, the protein molecular weight is 10638 Da, and theoretical pI:8.66, atomic composition: C461H734N140O136S7, extinction coefficients measured at 280 nm: 19605, the instability index: 75.26, aliphatic index: 61.35, grand average of hydropathicity: -0.899. This protein is same as Arabidopsis TRY protein and located in nucleolus. It belongs to instability, hydrophobic proteins and transcription factors of MYB family, and without signal peptide, glycosylation sites, trans-membrane structure, and coiled coil structure. Function prediction demonstrated that the protein coded by cotton TRY gene plays the same function as the protein does, coded by Arabidopsis TRY gene.
Cotton TRY gene was recombined to pBI121 vector.. And integrate the cotton TRY gene to tobacco genome by the ways of Agrobacterium tumefaciens mediating. Masculine transgene plant could be got by real time fluorescence PCR detection. It is initially indicated that cotton TRY gene possess the function to restrain the growth of epidermal hair cell by observing and analyzing the development of Masculine transgene tobacco leaf hair and roots hair.
引文
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